The Research On The Interactions Between STIM1and Orai1Signaling Molecules In Artery Smooth Muscle Cells

Objective：1．Research localization of protein STIM1and Orai1in arterial smooth muscle cells.2．Investigate the interactions between protein STIM1and Orai1in arterial smoothmuscle cells.Method：1．Conventionally culture rattus thoracic aorta smooth muscle cell A7r5.2．Use RT-PCR method to get gene Orai1and STIM1,construct the recombinantvector pEGFP-C₁-STIM1and pEGFP-C₁-Orai1,which expresses fused proteinSTIM1and Orai1,and construct the recombinant vector pIRES₂-EGFP-Orai1andpIRES₂-EGFP-STIM1which express proteins Orai1, STIM1and EGFPseparately.3．Use Western-blot method to detect the expression of Orai1and STIM1gene inA7r5cells.4．Use recombinant vectors pIRES₂-EGFP-Orai1and pIRES₂-EGFP-STIM1totransfect the A7r5cells, then use Western-blot to detect the Orai1and STIM1,andcompare the expression differences before and after transfection.5．Use recombinant vectors pEGFP-C₁-Orai1and pEGFP-C₁-STIM1to transfect A7r5cells seperately, then use laser confocal scanning microscope to localize theprotein STIM1and Orai1.6．Use immunofluorescence method to localize protein Orai1and STIM1in A7r5cells.7．Conventionally culture A7r5cells,CPA to stimulate cells for10minutes,extractprotein,then use Co-Immunoprecipitation method to identify the interactionsbetween STIM1and Orai1. 8．Use recombinant vectors pIRES₂-EGFP-Orai1and pIRES₂-EGFP-STIM1totransfect the A7r5cells, the use CPA to stimulate cells,and estract the protein,useCo-Immunoprecipitation method to identify the interactios between STIM1andOrai1.Result：1．Successfully constructed four kinds of recombinant vectors pEGFP-C₁-Orai1,pEGFP-C₁-STIM1, pIRES₂-EGFP-Orai1and pIRES₂-EGFP-STIM1.2．Used Western-blot method detected the expression of protein STIM1and Orai1inA7r5cells.3． Used pIRES₂-EGFP-Orai1and pIRES₂-EGFP-STIM1trafected A7r5cells,extracted proteins and detected the overexpression of protein STIM1and Orai1.4．Used pEGFP-C₁-STIM1transfect cells and immunofluorence methods, determinedthe STIM1protein localized at the endpllasmatic membrane.5．Conventionally cultured A7r5cells, used STIM1and Orai1antibody to doCo-Immunoprecipitation, determined the direct interactions between STIM1andOrai1.6．Used pIRES₂-EGFP-Orai1and pIRES₂-EGFP-STIM1trafect A7r5cells, andSTIM1and Orai1antibody to do Co-Immunoprecipitation, determined the directinteractions between STIM1and Orai1.Conclusion：1．Initially showed the STIM1protein localized at the endpllasmatic membrane inA7r5cells.2．Protein STIM1and Orai1interacts directly with each other in A7r5cells.